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Peroxisomes are present in virtually every eukaryotic cell type except the mature erythrocyte. In higher eukaryotes, one of the main functions of peroxisomes is the β-oxidation of very-long-chain fatty acids (VLCFA; > 22 carbon atoms) (1). The importance of peroxisomal β-oxidation is emphasized by the existence of a variety of different diseases in which peroxisomal β-oxidation is impaired and VLCFA concentrations are increased (1-4). Peroxisomal disorders can be categorized as (a) single peroxisomal enzyme deficiencies, including X-linked adrenoleukodystrophy (X-ALD) and disorders attributable to defects in one of the peroxisomal β-oxidation enzymes, such as acyl-CoA oxidase (AOX) deficiency and bifunctional protein (DBP) deficiency; and (b) disorders attributable to defects in peroxisome biogenesis. The peroxisome biogenesis disorders (PBDs) represent a continuum of clinical features ranging from the most severe form, Zellweger syndrome, through neonatal adrenoleukodystrophy to the least severe form, infantile Refsum disease.
Currently, measurement of the peroxisomal fatty acid β-oxidation activity is performed with 1-[^sup 14^C]-radiolabeled VLCFA substrates and one of two available methods: either in intact human skin fibroblasts cultured in monolayer (5); or in isolated fibroblasts permeabilized with digitonin (6). We investigated the feasibility of using deuterium-labeled tetracosanoic acid (D3-C24:0) as an alternative substrate to radiolabeled 1-[^sup 14^C]-labeled C24:0 for the measurement of peroxisomal β-oxidation activity in cultured primary human skin fibroblasts.
Before use, the purity of 24,24,24-D^sub 3^-C24:0 (Larodan Fine Chemicals AB) was determined. The D^sub 3^-C24:0 substrate contained ~6% deuterium-labeled octadecanoic acid (D^sub 3^-C18:0). Acetone was used to purify D^sub 3^-C24:0 according to the following procedure: 4 mL of acetone was added to 20 mg of D^sub 3^-C24:0. The sample was vortex-mixed vigorously, left at room temperature for 30 min, and centrifuged at 1600g for 10 min; approximately 80% of the acetone was then removed, and 3 mL of fresh acetone was added. This procedure was repeated two more times. After three washing steps with acetone, ~80% of the acetone was removed, and the remaining acetone was evaporated at room temperature under a constant stream of nitrogen. The residue was weighed, and a stock solution of 10 mmol/L D^sub 3^-C24:0 in absolute ethanol was prepared. After purification, the purity of D^sub 3^-C24:0 was analyzed, and the contribution of the D^sub 3^-C18:0 contaminant was determined to be <0.2%.
Fibroblasts from healthy controls and...